Ophthalmic compositions and methods for treating eyes

ABSTRACT

Ophthalmic compositions including compatible solute components and/or polyanionic components are useful in treating eyes, for example, to relieve dry eye syndrome, to protect the eyes against hypertonic insult and/or the adverse effects of cationic species on the ocular surfaces of eyes and/or to facilitate recovery from eye surgery.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/987,333, filed Jan. 4, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/248,227, filed Apr. 8, 2014, now U.S. Pat. No.9,254,324, issued Feb. 9, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/024,332, filed Sep. 11, 2013, now U.S. Pat. No.8,729,125, issued May 20, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/842,555, filed Mar. 15, 2013, now U.S. Pat. No.8,569,370, issued Oct. 29, 2013, which is a divisional of U.S. patentapplication Ser. No. 10/990,811, filed on Nov. 16, 2004, now U.S. Pat.No. 8,569,367, issued Oct. 29, 2013, the disclosures of which are herebyincorporated in their entirety by reference.

BACKGROUND OF THE INVENTION

The present invention relates to ophthalmic compositions and methodsuseful for treating eyes. More particularly, the present inventionrelates to ophthalmic compositions including mixtures of componentswhich are effective in providing desired protection to ocular surfacesof human or animal eyes, and to methods for treating human or animaleyes using ophthalmic compositions, for example, the present ophthalmiccompositions.

Mammalian eyes, such as human and other mammalian (animal) eyes,advantageously are adequately lubricated to provide eye comfort and tomore effectively provide good, clear vision. Ordinarily, suchlubrication is obtained naturally from a tear film, which is formed overthe outer, exposed ocular surface of the eye. This tear film is acomplex fluid that is normally continuously replenished by the lacrimal,meibomian, and other glands, and when intact provides essentialhydration and nutrients to the ocular surface. In addition to coatingand protecting the delicate ocular surface, the tear film/air interfacealso serves as the initial refractive surface of the eye. However, inmany instances, this tear film is not present in a sufficient amount,and a condition known as “dry eye” can result.

A relatively large number of compositions have been suggested for use inthe treatment and management of dry eye syndrome. For example,artificial tears, that is materials having chemical compositions whichmimic or resemble the functioning of natural tears, have been used. Suchartificial tears often require very frequent use since they are rapidlylost from the eye. In addition, although they wet the eye, their valuein lubricating the eye is somewhat less than desired. Compositions whichinclude specific lubricants have been suggested. For example, a numberof compositions including carboxy methylcelluloses (CMCs) have been usedin eyes.

Under normal conditions, the ocular surface of a human or animal eye isbathed in tears of a normal osmotic strength, for example, substantiallyisotonic. If this osmotic strength is increased, cells on the ocularsurface are exposed to a hyperosmotic or hypertonic environmentresulting in adverse reduction in cell volume due to trans-epithelialwater loss, and other undesired changes. The compensatory mechanisms arelimited, in many respects, leading to ocular surface compromise anddiscomfort. For example, the cells may attempt to balance osmoticpressure by increasing internal electrolyte concentration. However, atelevated electrolyte levels, cell metabolism is altered in many ways,including the reduction in enzyme activity and membrane damage. Inaddition, a hypertonic environment has been shown to be pro-inflammatoryto the ocular surface.

The cells of many life forms can compensate for hypertonic conditionsthrough the natural accumulation or manufacture of so-called “compatiblesolutes”, that work like electrolytes to balance osmotic pressure yet donot interfere with cellular metabolism like electrolytes. Compatiblesolutes or compatible solute agents, generally, are uncharged, can beheld within a living cell, for example, an ocular cell, are ofrelatively small molecular weight and are otherwise compatible with cellmetabolism. Compatible solutes are also considered to be osmoprotectantssince they may allow cell metabolism and/or enhance cell survival underhypertonic conditions that would otherwise be restricting.

For example, a class of organisms called halophiles exist that inhabithypersaline environments such as salt lakes, deep sea basins, andartificially-created evaporation ponds. These organisms may beeukaryotic or prokaryotic, and have mechanisms for synthesizing and/oraccumulating a variety of compatible solute agents, including polyols,sugars, and amino acids and their derivatives such as glycine, betaine,proline, ectoine, and the like.

Glycerin (glycerol) is a widely used osmotic agent that has beenidentified as a compatible solute in a variety of cells from a number ofdifferent species. It is also regarded as a humectant and ophthalmiclubricant. In the U.S., it is applied topically to the ocular surface torelieve irritation at concentrations up to 1%, and has been used athigher concentrations to impart osmotic strength in prescriptionmedications. Given its small size and biological origin, it shouldeasily cross cell membranes, and transport channels have been recentlyidentified in some cell types to facilitate glycerol movement.

Although glycerol may serve as the sole compatible solute, it may beexcessively mobile, that is, cross membranes too freely, to provide anextended benefit in certain systems. An example is the human tear filmwhere natural levels of glycerol are low. When a topical preparation isapplied, migration into the cell is likely to occur fairly rapidly.However, as concentration in the tear falls, glycerol may be lost overtime from cell to tear film, limiting the duration of benefit.

Another major class of compounds with osmoprotective properties in avariety of tissues is certain amino acids. In particular, betaine(trimethyl glycine) has been shown to be actively taken up by renalcells in response to osmotic challenge, and taurine is accumulated byocular cells under hypertonic conditions.

There continues to be a need to provide ophthalmic compositions, forexample, artificial tears, eye drops and the like, which are compatiblewith ocular surfaces of human or animal eyes and advantageously areeffective to allow such ocular surfaces to better tolerate hypertonicconditions.

Hypotonic compositions have been used on eyes as a method to counteractthe effects of hypertonic conditions. These compositions effectivelyflood the ocular surface with water, which rapidly enters cells whensupplied as a hypotonic artificial tear. Due to the rapid mobility ofwater into and out of cells, however, any benefit of a hypotoniccomposition will be extremely short-lived. Further, it has beendemonstrated that moving cells from a hypertonic environment to anisotonic or hypotonic environment down-regulates transport mechanismsfor cells to accumulate compatible solutes. Thus, use of a hypotonicartificial tear reduces the ability of cells to withstand hypertonicitywhen it returns shortly after drop instillation.

The clinical observation that agents such as carboxy methylcellulosesodium (CMC) and sodium hyaluronate (SH) are useful in treating signsand symptoms of dry eye syndrome or disease is well established. Thesetwo polyanionic agents have also been shown to be particularly useful inconditions where induced corneal compromise (CMC and LASIK surgicalprocedures) or allergic corneal insult (SH and shield ulcers in allergy)exist.

In addition, the tear film of the presumed normal human or animal eyemay have elevated (detectable) levels of Major Basic Protein (MBP)whereas it was previously believed that this protein was only expressedunder conditions of allergy with eosinophilic involvement (late phaseallergy). MBP is now recognized to be produced by Mast Cells (MC) aswell as eosinophils, which are known to commonly reside within ocularsurface tissues and are recognized to de-granulate, releasing MBP andother cationic compounds, under antigenic stimulation, mechanicaltrauma, and other conditions.

Another group of cationic proteins active on the ocular surface are oneor more of the defensins, which are normally part of the body'santimicrobial defense system. Defensins are found at increased levels inthe tear film of dry eye patients, and may either directly or throughinteraction with other substances have adverse effects on the health ofthe ocular surface.

There are recognized treatments designed to reduce the likelihood of MCde-granulation, most of which are used on the ocular surface inconjunction with treating seasonal or perennial allergic conjunctivitis.However, once de-granulation occurs, there are no recognized treatmentsto sorb, clear or deactivate released cationic mediators including MBP.Saline irrigation would dilute the agents but is impractical in mostcases. Also, recent data indicates that there is detectable MBP on theocular surface even in non-allergic eyes, meaning that an overabundanceof MBP and potential low-grade ocular surface damage may occur toindividuals at any given time.

It would be advantageous to provide ophthalmic compositions which areeffective to mitigate against or reduce the adverse effects of cationic,for example, polycationic, materials on ocular surfaces of human oranimal eyes.

SUMMARY OF THE INVENTION

New ophthalmic compositions for treating eyes, and methods of treatingeyes have been discovered. The present compositions very effectivelytreat eyes, for example, eyes afflicted or susceptible todiseases/conditions, such as, without limitation, dry eye syndrome, lowhumidity environments, and stress/trauma, for example, due to surgicalprocedures, and the like. In particular, these compositions would beuseful for mitigating the damaging effects of a hypertonic tear film,regardless of cause. The present compositions are relativelystraightforward, can be easily and cost effectively manufactured, andcan be administered, for example, topically administered, to an ocularsurface of an eye very conveniently.

In one broad aspect of the present invention, ophthalmic compositionsare provided comprising a carrier component, advantageously an aqueouscarrier component, and an effective amount of a tonicity componentincluding a material selected from compatible solute components, forexample, one or more of certain compatible solute agents, and mixturesthereof. In one very useful embodiment, the tonicity component comprisesa material selected from erythritol components and mixtures thereof. Inone additional embodiment, the tonicity component comprises a materialselected from combinations of at least two different compatible soluteagents.

In another broad aspect of the invention, ophthalmic compositions areprovided comprising a carrier, for example, an aqueous carrier,component, and an effective amount of a material selected from inositolcomponents, xylitol components and mixtures thereof. The osmolality ofsuch compositions are often higher or greater than isotonic, forexample, in a range of at least 310 to about 600 or about 1000mOsmols/kg.

In a further broad aspect of the invention, ophthalmic compositions areprovided which comprise a carrier, for example, an aqueous carrier,component, and an effective amount of a tonicity component comprising amaterial selected from carnitine components and mixtures thereof. In aparticularly useful embodiment, the composition has a non-isotonicosmolality.

In an additional aspect of the present invention, ophthalmiccompositions are provided which comprise a carrier, for example, anaqueous carrier, component, and an effective amount of a tonicitycomponent comprising a material selected from a mixture or combinationof compatible solute agents, for example, selected from mixtures of oneor more polyol components and/or one or more amino acid components.

In each of the above-noted aspects of the invention, the presentcompositions advantageously have chemical make-ups so as the material orthe mixture of organic compatible solute included in the tonicitycomponent is effective, when the composition is administered to an eye,to allow an ocular surface of the eye to better tolerate a hypertoniccondition on the ocular surface relative to an identical compositionwithout the material or the mixture of organic compatible solute agents.

A still further broad aspect of the invention provides ophthalmiccompositions comprising carrier component, a tonicity component and apolyanionic component. The tonicity component is present in an amounteffective to provide the composition with a desired osmolality, andcomprises a compatible solute component. The polyanionic component ispresent in an amount, when the composition is administered to a human oranimal eye, to reduce at least one adverse effect of a cationic, forexample, a polycationic, material on an ocular surface of a human oranimal eye relative to an identical composition without the polyanioniccomponent. This cationic material could be from any source, for example,may be endogenous, an environmental contaminant, or as an undesiredconsequence of applying an agent to the eye, for example a preservedsolution or contact lens care product. In one very useful embodiment,hyaluronic acid is not the sole polyanionic component. Other polyanioniccomponents are more suited for use in the present compositions, forexample, are more suited than hyaluronic acid or its salts for topicaladministration to an ocular surface of a human or animal eye. In anotherembodiment of the present invention, the composition has an osmolalityin a range of about 300 to about 600 or about 1000 mOsmols/kg.

One further broad aspect of the invention provides ophthalmiccompositions comprising a carrier component, and a polyanionic componentselected from polyanionic peptides, polyanionic peptide analogs,portions of polyanionic peptide analogs, carboxymethyl-substitutedpolymers of sugars, including but not limited to, glucose and the likesugars and mixtures thereof. Such polyanionic components are present inan amount effective, when the compositions are administered to a humanor animal eye, to reduce at least one adverse effect of a cationic, forexample, polycationic, species and/or substance on an ocular surface ofthe eye relative to an identical composition without the polyanioniccomponent.

Methods of treating human or animal eyes are also provided. Such methodscomprise administering a composition, for example, a composition inaccordance with the present invention, to a human or animal eye toprovide at least one benefit to the eye.

Any and all features described herein and combinations of such featuresare included within the scope of the present invention provided that thefeatures of any such combination are not mutually inconsistent.

These and other aspects of the present invention, are apparent in thefollowing detailed description, accompanying drawings, examples andclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical presentation of the intensity with regard tophosphorylated c-jun N-terminal kinases (p-JNK1 and p-JNK2) of certainophthalmic compositions.

FIG. 2 is a graphical presentation of the intensity with regard top-JNK1 and p-JNK2 of certain other ophthalmic compositions.

FIG. 3 is a graphical presentation of Phosphorylated:total JNK ratiosfor certain ophthalmic compositions obtained using the Beadlyte method.

FIG. 4 is a graphical presentation of Phospho:total p38 MAP Kinase forcertain ophthalmic compositions obtained using the Beadlyte method.

FIG. 5 is a graphical presentation of Phospho:total ERK MAP Kinase forcertain ophthalmic compositions obtained using the Beadlyte method.

FIG. 6 is a graphical presentation of a summary of concentrationdependent effects on trans-epithelial electrical resistance (TEER) forvarious ophthalmic compositions.

FIG. 7 is a graphical presentation of the effects on TEER of variousophthalmic compositions including compositions including combinations ofcompatible solute agents.

FIG. 8 is a graphical presentation of the effects on TEER of variousother ophthalmic compositions including compositions includingcombinations of compatible solute agents.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to ophthalmic compositions useful intreating human or animal eyes. As noted above, in one aspect of theinvention, compositions are provided which include a carrier component,for example, an aqueous-based or aqueous carrier component, and atonicity component comprising a material selected from at least onecompatible solute component, for example organic compatible solutecomponent. Such compositions advantageously include an effective amountof the material so that, when the composition is administered to an eye,the material is effective to allow an ocular surface of an eye to bettertolerate a hypertonic condition on the ocular surface relative to anidentical composition without the material.

Although such compositions may have any suitable tonicity or osmolality,for example, a hypotonic osmolality, a substantially isotonic osmolalityor a hypertonic osmolality, very useful compositions have osmolalitiesother than isotonic osmolality, for example, greater than isotonicosmolality. In one embodiment, the present compositions haveosmolalities in a range of at least about 300 or about 310 to about 600or about 1000 mOsmols/kg.

Polyols, such as erythritol components, xylitol components, inositolcomponents, and the like and mixtures thereof, are effectivetonicity/osmotic agents, and may be included, alone or in combinationwith glycerol and/or other compatible solute agents, in the presentcompositions. Without wishing to limit the invention to any particulartheory of operation, it is believed that because of their increasedsize, relative to glycerol, these polyol components when used topicallyon the eye, accumulate in the cells more slowly than glycerol, butremain within the cells for prolonged periods of time relative toglycerol.

In one very useful embodiment, mixtures of two or more differentcompatible solute components, for example, glycerol and/or one or moreother polyol components and/or one or more other compatible solutecomponents, for example, one or more uncharged or zwitterionic aminoacid components and the like, may be advantageously used together toprovide one or more benefits to the eye that are not obtained usingcompositions including only a single compatible solute component.

As used herein, the term “component” as used herein with reference to agiven compound refers to the compound itself, isomers and stereoisomers,if any, of the compound, suitable salts of the compound, derivatives ofthe compound and the like and mixtures thereof.

As use herein, the term “derivative” as it relates to a given compoundrefers to a compound having a chemical make-up or structure sufficientlysimilar to the given compound so as to function in a mannersubstantially similar to a substantially identical to the given compoundin the present compositions and/or methods.

Comfort and tolerability can be considered in formulating the presentcompositions. The amount of organic compatible solute component employedin the present compositions should be effective in providing at leastone benefit to the eye of a patient without unduly adversely affectingthe patient, for example, without unduly inducing discomfort, reflextearing and the like adverse affects.

For a formulator schooled in the art, it is possible to make thickfluids and gels that are retained for greater periods on the ocularsurface than thin fluids, with the trade-off often being a transientvision blur. Thick fluids and gels however have the advantage of lessfrequent dosing to deliver a given amount of substance.

Xylitol or erythritol used alone may require prolonged contact time toallow them to function effectively as a compatible solute component, forexample, due to the time needed for cellular uptake. However once insitu, for example, within ocular surface cells, the beneficial action ofbalancing hypertonic conditions advantageously is longer than with anequivalent amount of glycerol, which moves more quickly into and out ofcells. Such longer lasting benefit, and less frequent dosing, can beobtained without blurred vision.

In one embodiment, the present compositions include a combination ormixture of compatible solute agents, with each agent advantageouslybeing of different chemical type and/or having a different molecularsize and/or mobility. Small mobile agents offer rapid but short durationeffectiveness, e.g., protection from hypertonic insult, whereas largeless mobile agents offer delayed but longer lasting protectioneffectiveness.

Xylitol, erythritol and glycerol all have high hydroxyl groupconcentrations: one per carbon. Hydroxyl groups allow for greater waterbinding and increase compound solubility. In compositions for treatmentof dry eye syndrome, such high hydroxy group concentration may enhanceperformance of the composition by preventing water loss from thetissues.

Among the polyols, the 5-carbon xylitol, 4-carbon erythritol, and3-carbon glycerol are preferred for ophthalmic use. The 2-carbon form(ethylene glycol) is a well-known toxin and is not suitable. The6-carbon forms (mannitol, sorbitol, and related deoxy compounds) may beuseful in combination with the smaller molecules. In one embodiment,combinations of polyols with 3 to 6 carbons, and 1 and 2 carbon deoxyderivatives including, without limitation, isomers, stereo-isomers andthe like, as appropriate, may be useful in the present invention.

Uncharged or zwitterionic amino acids are useful as organic compatiblesolute components in accordance with the present invention.

Carnitine components, for example, carnitine itself,isomers/stereo-isomers thereof, salts thereof, derivatives thereof andthe like and mixtures thereof, are very useful compatible solutecomponents for use in the present ophthalmic compositions. Carnitine iswell-established as necessary for various parts of fatty acidmetabolism, so it has a significant role in the metabolism of liver andmuscle cells. Carnitine may serve as an energy source for many types ofcells, including ocular cells. Carnitine components may have uniqueproperties in multiple roles, for example as osmoprotectants, in fattyacid metabolism, as an antioxidant, in promoting wound healing, as aprotein chaperone, and in neuroprotection.

The organic compatible solute component may be advantageously providedin the present compositions by using a combination of such agents ormaterials of differing size, mobility, and mechanism of action. Smallmobile agents, such as smaller polyols, would be predicted to offerrapid but short duration osmoprotection. Several of the amino acids andrelated compounds may function as long-acting intracellular compatiblesolutes and protein stabilizers. In the present invention, carnitinecomponents may be used alone or in combination with one or more otheramino organic compatible solute components and/or polyols, for example,as described herein.

Amine-based organic compatible solute components and/or components thatmay be used include, but are not limited to, betaine, taurine,carnitine, sarcosine, proline, trimethylamines in general, otherzwitterionic amino acids and the like and mixtures thereof. Polyols thatmay be useful in combination with carnitine and/or one of the otheramine-based organic compatible solute components include, but are notlimited to, glycerol, propylene glycol, erythritol, xylitol,myo-inositol, mannitol, sorbitol and the like and mixtures thereof.

The amount of the compatible solute component included in the presentcompositions may be any suitable amount. However, such amountadvantageously is effective to provide a benefit to the eye as a resultof the administration of the composition containing the compatiblesolute component to the eye. Excessive amounts of compatible solutecomponents are to be avoided, since such amounts can cause discomfort tothe patient and/or potential harm to the eye being treated. Thecompatible solute component advantageously is present in an amounteffective in providing the desired osmolality to the composition.

The specific amount of compatible solute component employed may varyover a wide range depending, for example, on the overall chemicalmake-up and intended use of the composition, on the desired osmolalityof the composition, on the specific compatible solute or combination ofsuch solutes being employed and the like factors. In one embodiment, thetotal amount of compatible solute component included in the presentcompositions may be in a range of about 0.01% (w/v) or about 0.05% (w/v)to about 1% (w/v) or about 2% (w/v) or about 3% (w/v) or more.

Corneal surface cells respond to osmotic forces by regulating salt andwater transport in an effort to maintain a constant cell volume. Inconditions of chronic hypertonicity, for example, such as exist in dryeye disease, transport mechanisms for uptake of compatible solutes,including various amino acids and polyols, are up-regulated. In oneembodiment of the present invention, ophthalmic compositions, forexample, artificial tears, containing a compatible solute component areformulated to have a tonicity higher or in excess of isotonicity,advantageously in a tonicity range of about 300 or about 310 to about600 or about 1000 mOsmols/kg. Without wishing to limit the invention toany particular theory of operation, it is believed that, under suchconditions, both immediate and long-term mechanisms to accumulatecompatible solutes in cells are stimulated, allowing enhanced uptake andretention compared to cellular activity under isotonic or hypotonicconditions. Once the compatible solute component is accumulated by thecells, the cells have enhanced protection from ongoing hypertonicinsult, for example, caused by dry eye syndrome and/or one or more otherconditions/diseases. Results of this enhanced protection includeimproved cellular metabolism and survival for a period of hours to daysfollowing application of an ophthalmic composition of the presentinvention.

In the normal lacrimal system, tear production, tear drainage, and tearevaporation is balanced in order to provide a moist, lubricated ocularsurface. Typical values for tear osmolarity range from 290 to 310mOsmols/kg in normal individuals, and these may change throughout theday or in response to changing environmental conditions. In the normalindividual, neural feedback from the ocular surface to the lacrimalglands controls tear production in order to maintain a stable ocularsurface fluid. It has been proposed that tear film tonicity is one ofthe principal stimuli for this regulatory feedback. In dry eye disease,dysfunction of the production apparatus (the various glands), thedrainage system, the neural signaling mechanism, or the ocular surfaceitself leads to an inadequate tear film, ocular surface compromise, andsubjective discomfort.

On the cellular level, dry eye disease is usually characterized by achronically hypertonic extracellular (tear film) environment. Publishedreports of the tonicity of the tear film of dry eye patients gives arange of 300 to 500 mOsmols/kg, with most values between 320 and 400mOsmols/kg. Under these conditions, cells will tend to lose water and/orgain salts, and may undergo cell volume changes. Hypertonicity has beenshown to alter cellular metabolic processes, reduce the functioning ofenzymatic processes, and lead to apoptosis and cell death.

As a defense against hypertonic challenge, corneal cells have beendemonstrated to up-regulate transport mechanisms for non-ionic solutessuch as amino acids and polyols, and accumulate these solutesintracellularly in order to maintain cell volume without changingelectrolyte balance. Under these conditions, cellular metabolism is lessaffected than with volume and electrolyte changes, and such compoundsare referred to as compatible solutes. Compatible solutes include butare not limited to the amino acids betaine (trimethylglycine), taurine,glycine, and proline, and the polyols glycerol, erythritol, xylitol,sorbitol, and mannitol. Compatible solutes are also considered to beosmoprotectants since they may allow cell metabolism or enhance cellsurvival under hypertonic conditions that would otherwise berestricting.

Cells accumulate certain compatible solutes by biosynthesis within thecell and others by increased trans-membrane transport from theextracellular fluid (in this case the tear fluid). In both cases,specific synthetic or transport proteins are involved in this process.Experimental evidence indicates that these proteins are activated in thepresence of hypertonic conditions, and that transcription andtranslation events to produce these proteins are up-regulated byhypertonic conditions. Conversely, experimental evidence indicates thatcorneal and other cells will expel compatible solutes when exposed tohypotonic conditions, or when moving from a hypertonic to an isotonicenvironment.

In dry eye disease, corneal surface cells are exposed to a hypertonicenvironment, and are stimulated to accumulate osmoprotectant substancesas they are available. The addition of an iso- or hypo-tonic artificialtear to the ocular surface provides relief from symptoms due to enhancedlubrication, but tends to down-regulate mechanisms in these cells foraccumulation of osmoprotectants. This may result in furthervulnerability to osmotic insult in the minutes to hours following dropuse as the tear film returns to its hypertonic dry eye state.

Current FDA guidance stipulates that “an ophthalmic solution should havean osmotic equivalence between 0.8 and 1.0 percent sodium chloride tocomply with labeling claims of ‘isotonic solution’.” This is equivalentto a range from 274 to 342 mOsm/kg. Further, FDA guidelines state that“two to 5 percent sodium chloride ophthalmic preparations are hypertonicand are acceptable OTC products when labeled as ‘hypertonic solutions’.”This range equates to 684 to 1711 mOsm/kg. For the purposes of thepresent invention, a “supra-tonic” solution is defined to have anosmolality intermediate between these two ranges, or approximately 300or 310 to about 600 or about 800 or about 1000 mOsmols/kg, equivalent toabout 0.9 to about 1.8 percent sodium chloride (1.8% is the maximum FDAguidance for topical ophthalmic solutions not labeled as hypertonic).

The present invention takes these concepts into account by formulatingan artificial tear at supra-tonic levels more compatible with theexisting hypertonic state of the dry eye ocular surface. In addition tobeing formulated in the supra-tonic range (about 300 or about 310 toabout 600 or about 1000 mOsmols/kg total tonicity), the presentcompositions contain one or more organic compatible solute agents asdescribed herein. The combination of supra-tonicity and inclusion of oneor more compatible solutes in the present compositions serve to bothstimulate or maintain uptake of these protective substances into thecorneal surface cells, and to provide abundant supplies of thesematerials or substances.

In addition to sufficient quantities of compatible solutes in asupra-tonic medium, the present compositions also may containappropriate demulcents and viscosity agents, which provide comfort andlubrication, and also advantageously are effective in holding theorganic compatible solute composition on the ocular surface forsufficient time to enhance uptake by the corneal surface cells.

It should be noted that FDA guidelines clearly indicate that the finaltonicity of the formulation may be determined by nonionic as well asionic species. Thus, the formula may contain significant amounts ofglycerol and other compatible solutes, and not contain substantialamounts or any of ionic tonicity agents, such as sodium salts. In oneembodiment, the present components are substantially free of ionictonicity agents.

Advantageously, the present compositions include a combination ofdifferent organic compatible solute agents effective to provide foruptake by corneal cells during the time of exposure to the drop duringuse, for example, about 5 to about 30 minutes, depending on viscosity,after administration, and to provide for intracellular retention duringthe period of hours between drop applications.

Because of the enhanced protection from osmotic insult provided by thepresent composition, the duration of clinical benefit resulting fromeach dosage or application is increased. With regular use of the presentcompositions, ocular surface health is enhanced as cells are lessmetabolically challenged and cell survival is enhanced.

Another aspect of the present invention, compositions comprising acarrier component and a polyanionic component are provided. Suchpolyanionic component-containing compositions advantageously, althoughin certain embodiments not necessarily, include organic compatiblesolute components as described herein.

In one embodiment, compositions are provided which comprise a carriercomponent and a polyanionic component in an amount effective to treat anocular surface of an eye under a condition of an increased population ofcationic species, for example and without limitation, increased MajorBasic Protein (MBP), and/or decreased polyanionic species on thesurface. In one embodiment, the present ophthalmic compositions includepolyanionic components present in amounts effective, when thecompositions are administered to human or animal eyes, to reduce atleast one adverse effect of a cationic, e.g., polycationic, material onan ocular surface relative to an identical composition without thepolyanionic component.

In one useful embodiment, compositions comprising polyanioniccomponents, for example, with or without the compatible solutecomponents, may be effectively used before, during and/or after surgicalprocedures, including without limitation, surgical procedures in whichthe eye is exposed to laser energy, for example, in the treatment ofpost-LASIK staining, dryness and other ocular surface complications. Theetiology of post-LASIK surface compromise may be multifactorial,including, without limitation, surgically-induced neurotrophichypesthesia and keratitis, damage to limbal cells from force of thesuction ring, altered lid apposition in blinking due to altered cornealtopography, chemical damage to ocular surface from topical medicationsand preservatives and the like.

The administration of polyanionic component-containing compositions, inaccordance with the present invention, to the ocular surface and tearfilm may be effective in treating one or more or even all, of the abovenamed causes of post-LASIK ocular surface compromise.

In one particularly useful embodiment, the present compositions includepolyanionic components that mimic the activity, for example, theantigenic and/or cytotoxic activity, of the pro-piece of MBP, which hasbeen shown to consist of a 90-residue polypeptide. Useful agents mayinclude one or more polypeptide analogs of this sequence or portions ofthis sequence.

As used herein, the term “mimic” means that the polyanionic component,e.g., polypeptide analog, has an activity within (plus or minus) about5% or about 10% or about 15% or about 20% of the corresponding activityof the pro-piece of MBP.

The pro-piece of MBP has an amino acid sequence as shown in SEQ ID NO:1below:

IhIrsetstf etplgaktlp edeetpeqem eetperelee eeewgsgsed

askkdgaves isvpdmvdkn ltcpeeedtv kvvgipgcq

A polypeptide analog of the Major Basic Protein pro-piece sequence or ofa portion of the Major Basic Protein pro-piece sequence means a peptidecomprising an amino acid sequence having at least about 75% or about 80%or about 85% or about 90% or about 95% or about 99% or more identity toa homologous continuous amino acid sequence comprised in SEQ ID NO:1, orportions thereof.

Carboxymethyl-substituted polymers of sugars, for example and withoutlimitation, glucose and the like sugars, may be employed as polyanioniccomponents in accordance with the present invention.

Further, additional useful polyanionic components include, withoutlimitation, modified carbohydrates, other polyanionic polymers, forexample, and without limitation, those already available forpharmaceutical use, and mixtures thereof. Mixtures of one or more of theabove-noted polypeptide analogs and one or more of the above-noted otherpolyanionic components may be employed.

The present compositions are advantageously ophthalmically acceptable,comprising an ophthalmically acceptable carrier component, a compatiblesolute component and/or a polyanionic component.

A composition, carrier component or other component or material is“ophthalmically acceptable” when it is compatible with ocular tissue,that is, it does not cause significant or undue detrimental effects whenbrought into contact with ocular tissue. Preferably, the ophthalmicallyacceptable component or material is also compatible with othercomponents of the present compositions.

As used herein, the term “polyanionic component” refers to a chemicalentity, for example, an ionically charged species, such as an ionicallycharged polymeric material, which includes more than one discreteanionic charge, that is multiple discrete anionic charges. Preferably,the polyanionic component is selected from the group consisting ofpolymeric materials having multiple anionic charges and mixturesthereof.

The polyanionic component may have a substantially constant or uniformmolecular weight, or may be made up of two or more polyanionic componentportions of different molecular weights. Ophthalmic compositions havingpolyanionic components including two or more portions of differentmolecular weights are disclosed in U.S. patent application Ser. No.10/017,817, filed Dec. 14, 2001, the disclosure of which is herebyincorporated in its entirety herein by reference.

Preferably, the composition has an increased ability to adhere to an eyewhen the composition is administered to an eye relative to asubstantially identical composition without the polyanionic component.With regard to the increased ability to adhere to an eye feature notedabove, the present compositions preferably are effective to provideeffective lubrication over a longer period of time before requiringreadministration relative to a substantially identical compositionwithout the polyanionic component.

Any suitable polyanionic component may be employed in accordance withthe present invention provided that it functions as described herein andhas no substantial detrimental effect on the composition as a whole oron the eye to which the composition is administered. The polyanioniccomponent is preferably ophthalmically acceptable at the concentrationsused. The polyanionic component preferably includes three (3) or moreanionic (or negative) charges. In the event that the polyanioniccomponent is a polymeric material, it is preferred that many of therepeating units of the polymeric material include a discrete anioniccharge. Particularly useful anionic components are those which are watersoluble, for example, soluble at the concentrations used in the presentcompositions at ambient (room) temperature.

Examples of suitable polyanionic components useful in the presentcompositions include, without limitation, anionic cellulose derivatives,anionic acrylic acid-containing polymers, anionic methacrylicacid-containing polymers, anionic amino acid-containing polymers andmixtures thereof. Anionic cellulose derivatives are very useful in thepresent invention.

A particularly useful class of polyanionic components are one or morepolymeric materials having multiple anionic charges. Examples include,but are not limited to:

metal carboxy methylcelluloses

metal carboxy methylhydroxyethylcelluloses

metal carboxy methylstarches

metal carboxy methylhydroxyethylstarches

metal carboxy methylpropyl guars

hydrolyzed polyacrylam ides and polyacrylonitriles

heparin

glycoam inoglycans

hyaluronic acid

chondroitin sulfate

dermatan sulfate

peptides and polypeptides

alginic acid

metal alginates

homopolymers and copolymers of one or more of:

-   -   acrylic and methacrylic acids    -   metal acrylates and methacrylates    -   vinylsulfonic acid    -   metal vinylsulfonate    -   amino acids, such as aspartic acid, glutamic acid and the like    -   metal salts of amino acids    -   p-styrenesulfonic acid    -   metal p-styrenesulfonate    -   2-methacryloyloxyethylsulfonic acids    -   metal 2-methacryloyloxethylsulfonates    -   3-methacryloyloxy-2-hydroxypropylsulfonic acids    -   metal 3-methacryloyloxy-2-hydroxypropylsulfonates    -   2-acrylamido-2-methylpropanesulfonic acids    -   metal 2-acrylamido-2-methylpropanesulfonates    -   allylsulfonic acid    -   metal allylsulfonate and the like.

Excellent results are achieved using polyanionic components selectedfrom carboxy methylcelluloses and mixtures thereof, for example, alkalimetal and/or alkaline earth metal carboxy methylcelluloses.

The present compositions preferably are solutions, although other forms,such as ointments, gels, and the like, may be employed.

The carrier component is ophthalmically acceptable and may include oneor more components which are effective in providing such ophthalmicacceptability and/or otherwise benefiting the composition and/or the eyeto which the composition is administered and/or the patient whose eye isbeing treated. Advantageously, the carrier component is aqueous-based,for example, comprising a major amount that is at least about 50% byweight, of water. Other components which may be included in the carriercomponents include, without limitation, buffer components, tonicitycomponents, preservative components, pH adjustors, components commonlyfound in artificial tears and the like and mixtures thereof.

The present compositions preferably have viscosities in excess of theviscosity of water. In one embodiment, the viscosity of the presentcompositions is at least about 10 cps (centipoise), more preferably in arange of about 10 cps to about 500 cps or about 1,000 cps.Advantageously, the viscosity of the present composition is in a rangeof about 15 cps or about 30 cps or about 70 to about 150 cps or about200 cps or about 300 cps or about 500 cps. The viscosity of the presentcomposition may be measured in any suitable, for example, conventionalmanner. A conventional Brookfield viscometer measures such viscosities.

In one very useful embodiment, the polyanionic component is present inan amount in a range of about 0.1% to about 5%, preferably about 0.2% toabout 2.5%, more preferably about 0.2% to about 1.8% and still morepreferably about 0.4% to about 1.3% (w/v) of the composition.

Other components which may be included in the carrier componentsinclude, without limitation, buffer components, tonicity components,preservative-components, pH adjustors, components commonly found inartificial tears, such as one or more electrolytes, and the like andmixtures thereof. In one very useful embodiment the carrier componentincludes at least one of the following: an effective amount of a buffercomponent; an effective amount of a tonicity component; an effectiveamount of a preservative component; and water.

These additional components preferably are ophthalmically acceptable andcan be chosen from materials which are conventionally employed inophthalmic compositions, for example, compositions used to treat eyesafflicted with dry eye syndrome, artificial tear formulations and thelike.

Acceptable effective concentrations for these additional components inthe compositions of the invention are readily apparent to the skilledpractitioner.

The carrier component preferably includes an effective amount of atonicity adjusting component to provide the composition with the desiredtonicity. The carrier component preferably includes a buffer componentwhich is present in an amount effective to maintain the pH of thecomposition in the desired range. Among the suitable tonicity adjustingcomponents that may be employed are those conventionally used inophthalmic compositions, such as one or more various inorganic salts andthe like. Sodium chloride, potassium chloride, mannitol, dextrose,glycerin, propylene glycol and the like and mixtures thereof are veryuseful tonicity adjusting components. Among the suitable buffercomponents or buffering agents that may be employed are thoseconventionally used in ophthalmic compositions. The buffer salts includealkali metal, alkaline earth metal and/or ammonium salts, as well ascitrate, phosphate, borate, lactate and the like salts and mixturesthereof. Conventional organic buffers, such as Goode's buffer and thelike, may also be employed.

Any suitable preservative component may be included in the presentcompositions provided that such components is effective as apreservative in the presence of the polyanionic component. Thus, it isimportant that the preservative component be substantially unaffected bythe presence of the polyanionic component. Of course, the preservativecomponent chosen depends on various factors, for example, the specificpolyanionic component present, the other components present in thecomposition, etc. Examples of the useful preservative componentsinclude, but are not limited to, per-salts, such as perborates,percarbonates and the like; peroxides, such as very low concentrations,e.g., about 50 to about 200 ppm (w/v), of hydrogen peroxide and thelike; alcohols, such as benzyl alcohol, chlorobutanol and like; sorbicacid and ophthalmically acceptable salts thereof and mixtures thereof.

The amount of preservative component included in the presentcompositions containing such a component varies over a relatively widerange depending, for example, on the specific preservative componentemployed. The amount of such component preferably is in the range ofabout 0.000001% to about 0.05% or more (w/v) of the present composition.

One particularly useful class of preservative components are chlorinedioxide precursors. Specific examples of chlorine dioxide precursorsinclude stabilized chlorine dioxide (SCD), metal chlorites, such asalkali metal and alkaline earth metal chlorites, and the like andmixtures thereof. Technical grade sodium chlorite is a very usefulchlorine dioxide precursor. Chlorine dioxide-containing complexes, suchas complexes of chlorine dioxide with carbonate, chlorine dioxide withbicarbonate and mixtures thereof are also included as chlorine dioxideprecursors. The exact chemical composition of many chlorine dioxideprecursors, for example, SCD and the chlorine dioxide complexes, is notcompletely understood. The manufacture or production of certain chlorinedioxide precursors is described in McNicholas U.S. Pat. No. 3,278,447,which is incorporated in its entirety herein by reference. Specificexamples of useful SCD products include that sold under the trademarkPurite® by Allergan, Inc., that sold under the trademark Dura Klor byRio Linda Chemical Company, Inc., and that sold under the trademarkAnthium Dioxide by International Dioxide, Inc.

The chlorine dioxide precursor is included in the present compositionsto effectively preserve the compositions. Such effective preservingconcentrations preferably are in the range of about 0.0002 or about0.002 to about 0.02% (w/v) or higher of the present compositions.

In the event that chlorine dioxide precursors are employed aspreservative components, the compositions preferably have an osmolalityof at least about 200 mOsmol/kg and are buffered to maintain the pHwithin an acceptable physiological range, for example, a range of about6 to about 8 or about 10.

The present compositions preferably include an effective amount of anelectrolyte component, that is one or more electrolytes, for example,such as is found in natural tears and artificial tear formulations.Examples of particularly useful such electrolytes for inclusion in thepresent compositions include, without limitation, alkaline earth metalsalts, such as alkaline earth metal inorganic salts, and mixturesthereof, e.g., calcium salts, magnesium salts and mixtures thereof. Verygood results are obtained using an electrolyte component selected fromcalcium chloride, magnesium chloride and mixtures thereof.

The amount or concentration of such electrolyte component in the presentcompositions can vary widely and depends on various factors, forexample, the specific electrolyte component being employed, the specificcomposition in which the electrolyte is to be included and the likefactors. In one useful embodiment, the amount of the electrolytecomponent is chosen to at least partially resemble, or evensubstantially resemble, the electrolyte concentration in natural humantears. Preferably, the concentration of the electrolyte component is inthe range of about 0.01 to about 0.5 or about 1% of the presentcomposition.

The present compositions may be prepared using conventional proceduresand techniques. For example, the present compositions can be prepared byblending the components together, such as in one bulk.

To illustrate, in one embodiment, the polyanionic component portions arecombined with purified water and caused to disperse in the purifiedwater, for example, by mixing and/or agitation. The other components,such as the buffer component, tonicity component, electrolyte component,preservative component and the like, are introduced as the mixingcontinues. The final mixture is sterilized, such as steam sterilized,for example, at temperatures of at least about 100° C., such as in arange of about 120° C. to about 130° C., for a time of at least about 15minutes or at least about 30 minutes, such as in a range of about 45 toabout 60 minutes. In one embodiment, the preservative componentpreferably is added to the mixture after sterilization. The finalproduct preferably is filtered, for example, through a 20 micronsterilized cartridge filter, such as a 20 micron clarity filtercartridge, e.g., sold by Pall under the tradename HDC II, to provide aclear, smooth solution, which is then aseptically filled intocontainers, for example, low density polyethylene teal containers.

Alternately, each of the polyanionic component portions can be mixedwith purified water to obtain individual polyanionic component portionsolutions. By mixing the individual polyanionic component portionsolutions together, a blend is easily and effectively obtained havingthe desired, controlled ratio of the individual polyanionic componentportions. The blended solution can then be combined with the othercomponents, sterilized and filled into containers, as noted above.

In one particularly useful embodiment, a solution of the polyanioniccomponent portions and purified water is obtained, as noted above. Thissolution is then sterilized, for example, as noted above. Separately,the other components to be included in the final composition aresolubilized in purified water. This latter solution is sterile filtered,for example, through a 0.2 micron sterilizing filter, such as that soldby Pall under the tradename Suporflow, into the polyanioniccomponent-containing solution to form the final solution. The finalsolution is filtered, for example, as noted above, to provide a clear,smooth solution which is then aseptically filled into containers, asnoted above.

The present compositions may be effectively used, as needed, by methodswhich comprise administering an effective amount of the composition toan eye in need of lubrication, for example, an eye afflicted with dryeye syndrome or having a propensity toward dry eye syndrome. Theadministering step may be repeated as needed to provide effectivelubrication to such eye. The mode of administration of the presentcomposition depends on the form of the composition. For example, if thecomposition is a solution, drops of the composition may be applied tothe eye, e.g., from a conventional eye dropper. In general, the presentcompositions may be applied to the surface of the eye in substantiallythe same way as conventional ophthalmic compositions are applied. Suchadministration of the present compositions does provide substantial andunexpected benefits, as described elsewhere herein.

The following non-limiting examples illustrate certain aspects of thepresent invention.

EXAMPLE 1

In this experiment, corneal epithelial cells were isolated from therabbit eye and grown under conditions so that they differentiate into alayered “air-lift” culture that includes basal, wing, and squamouscells. As they grow and differentiate, these cultures developed tightjunctions between cells that provide the basis for a trans-epithelialelectrical resistance (TEER) across the cell layers between the apicaland basal surfaces. The TEER value is a sensitive measure of cellgrowth, differentiation and health.

After 5 days in culture during which the layered structure forms,different culture wells were exposed to hypertonic fluid (400mOsmols/kg) with or without addition of one of 6 candidate compatiblesolutes at a low concentration (2 mM). The TEER was then measured after22 hours of exposure. The TEER value was expressed as a percentage ofthe TEER value obtained from a similar culture under isotonic (300mOsmol/kg) conditions. The results of these tests are shown in Table 1.

TABLE 1 Test Results TEER (as % of isotonic Compatible Solute control)at 22 hours Isotonic Control 100% Hypertonic Control 23.3 2 mM Taurine39.8 2 mM Betaine 53.3 2 mM Carnitine 118.9 2 mM Erythritol 107.4 2 mMMyo-Inositol 74.8 2 mM Xylitol 94.1

These results demonstrate that all of the candidates tested have someosmoprotective ability, increasing the TEER relative to the hypertoniccontrol. Surprisingly, of the agents tested, carnitine produced the mostbenefit. Without wishing to limit the invention to any particular theoryof operation, it is believed that the beneficial results obtained withcarnitine may relate to carnitine's multiple roles in energy metabolismand other cellular mechanisms as well as its osmoprotective effects.

Further, and also unexpectedly, erythritol provided the best resultsamong the polyols tested. Xylitol and myo-inositol provided goodresults.

These results indicate that each of the 6 candidate compounds, andpreferably, carnitine, erythritol, xylitol and myo-inositol, may beuseful in ophthalmic compositions, for example, to mitigate againsthypertonic conditions on ocular surfaces of human or animal eyes.

Again, without wishing to limit the invention to any particular theoryof operation, it is believed that, due to the varying roles a number ofthese compounds may play, that combinations of 2 or more of thesecompounds, for example, including at least one polyol and at least oneamino acid, are likely to provide increased protection of cornealsurfaces from insults, for example, due to desiccation andhyperosmolality, such as occur in dry eye disease.

EXAMPLE 2

Phosphorylated JNK (the activated form of the stress associated proteinkinase, SAPK) plays a key role in induction of inflammation andapoptosis in response to stress, including hyperosmolarity.

Human corneoscleral tissues, from donors aged 16-59 years were obtainedfrom the Lions Eye Bank of Texas (Houston, Tex.). Corneal epithelialcells were grown from limbal explants. In brief, after carefullyremoving the central cornea, excess conjunctiva and iris and cornealendothelium, the limbal rim was cut into 12 equal pieces (about 2×2 mmsize each). Two of these pieces were placed epithelial side up into eachwell of 6-well culture plates, and each explant was covered with a dropof fetal bovine serum (FBS) overnight. The explants were then culturedin SHEM medium, which was an 1:1 mixture of Dulbecco modified Eaglemedium (DMEM) and Ham F-12 medium containing 5 ng/mL EGF, 5 μg/mLinsulin, 5 μg/mL transferrin, 5 ng/mL sodium selenite, 0.5 μg/mLhydrocortisone, 30 ng/mL cholera toxin A, 0.5% DMSO, 50 μg/mLgentamicin, 1.25 μg/mL amphotericin B and 5% FBS, at 37° C. under 5% CO₂and 95% humidity. The medium was renewed every 2-3 days. Epithelialphenotype of these cultures was confirmed by characteristic morphologyand immuno-fluorescent staining with cytokeratin antibodies (AE-1/AE-3).

Cell culture dishes, plates, centrifuge tubes and other plastic warewere purchased from Becton Dickinson (Lincoln Park, N.J.). Dulbeccomodified Eagle medium (DMEM), Ham F-12 medium, Fungizone, and gentamicinwere from Invitrogen-GIBCO BRL (Grand Island, N.Y.). Fetal bovine serum(FBS) was from Hyclone (Logan, Utah).

A series of primary sub-confluent corneal epithelial cultures (grown for12 to 14 days, about 4-5×10⁵ cells/well) were washed three times withpreserved buffered saline (PBS) and switched to an Earle's Balanced SaltSolution (EBSS, 300 mOsmols/kg) for 24 hours before treatment. Thecorneal epithelial cells were cultured for 1 hour in an equal volume(2.0 mL/well) of EBSS media or 400 mOsmols/kg media by adding 53 mM NaClor sucrose, with either L-carnitine inner salt, betaine hydrochloride,erythritol, or xylitol (all at a concentration of 2 mM) that werepre-added 60 minutes before adding NaCl or sucrose. Samples withoutthese osmoprotectants were also prepared and tested.

The adherent cells were lysed in Beadlyte® Buffer B (included in theBeadlyte® Cell Signaling buffer kit, Upstate Biotechnology, Lake Placid,N.Y.) containing an EDTA-free protease inhibitor cocktail tablet (RocheApplied Science, Indianapolis, Ind.) for 15 minutes. The cell extractswere centrifuged at 12,000×g for 15 minutes at room temperature and thesupernatants were stored at −80° C. until they were analyzed by Westernblot analysis. The total protein concentrations of the cell extractswere determined using a Micro BCA protein assay kit (Pierce, Rockford,Ill.).

The intensity of each of JNK1 and JNK2 was tested for each of thesecompositions using Western blot analysis with specific antibodies toeach phosphorylated species.

The Western blot analysis was conducted as follows. The protein samples(50 μg per lane) were mixed with 6×SDS reducing sample buffer and boiledfor 5 minutes before loading. Proteins were separated by SDSpolyacrylamide gel electrophoresis (4-15% Tris-HCl, gradient gels fromBio-Rad, Hercules, Calif.), and transferred electronically topolyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.).The membranes were blocked with 5% non-fat milk in TTBS (50 mM Tris, pH7.5, 0.9% NaCl, and 0.1% Tween-20) for 1 hour at room temperature (RT),and then incubated 2 hours at RT with a 1:1000 dilution of rabbitantibody against phospho-p38 MAPK (Cell Signaling, Beverly, Mass.),1:100 dilution of rabbit antibody against phospho-JNK, or 1:500 dilutionof monoclonal antibody against phospho-p44/42 ERK (Santa CruzBiotechnology, Santa Cruz, Calif.).

After three washings with TTBS, the membranes were incubated for 1 hourat RT with horseradish peroxidase-conjugated secondary antibody goatanti-rabbit IgG (1:2000 dilution, Cell Signaling, Beverly, Mass.), orgoat anti-mouse IgG (1:5000 dilution, Pierce, Rockford, Ill.). Afterwashing the membranes four times, the signals were detected with an ECLadvance chemiluminescence reagent (Amersham, Piscataway, N.J.) and theimages were acquired by a Kodak image station 2000R (Eastman Kodak, NewHaven, Conn.). The membranes were stripped in 62.5 mM Tris HCl, pH 6.8,containing 2% SDS and 100 mM α-mercaptoethanol at 60° C. for 30 minutes,then they were re-probed with 1:100 dilution of rabbit antibody againstJNK (Santa Cruz Biotechnology) or 1:1000 dilution of rabbit antibodiesagainst ERK or p38 MAPK (Cell Signaling). These three antibodies detectboth phosphorylated and un-phosphorylated forms which represent thetotal levels of these MAPKs. The signals were detected and captured asdescribed above.

An intensity score is determined from image analysis of the resultingbands.

Test results are shown in FIGS. 1 and 2.

Referring now to FIG. 1, there was no effect on JNK activation witheither erythritol or xylitol. However, with reference to FIG. 2, therewas a definite decrease in the levels of JNK1 and JNK2 in L-carnitineand betaine cultures compared to 400 mOsmols/kg media alone. There wasalso a less robust effect in the 300 mOsmols/kg cultures.

EXAMPLE 3

In another series of experiments, the Beadlyte® Cell Signaling Assay wasused. This assay is a fluorescent bead-based sandwich immunoassay. Eachsample (10 μg/25 μL) was pipetted into a well of a 96-well plate andincubated with 25 μL of diluted 5×beads coupled to phospho-JNK,phospho-ERK, phospho-p38 or total JNK, or total ERK, or total p38specific capture antibodies overnight. Overnight incubation was utilizedfor the reaction of the capture beads with the proteins from the celllysates.

The beads were washed and mixed with biotinylated specific reporterantibodies for phospho-MAPK or total-MAPK, followed bystreptavidin-phycoerythrin. The amount of total or phospho-MAPK was thenquantified by the Luminex 100™ system (Luminex, Austin, Tex.). Fiftyevents per bead were read, and the data output obtained from theBio-Plex Manager software were exported to Microsoft Excel® for furtheranalysis. The results were presented as the percentage of phospho-MAPKto total-MAPK.

Results of these tests are shown in FIGS. 3, 4 and 5.

As shown in FIG. 3, all of the candidate materials, that is, all oferythritol, xylitol, L-carnitine and betaine, reduced the amount ofphospho-total JNK relative to the hypertonic control.

With reference to FIG. 4, all of the candidate materials, with theexception of betaine, reduced the amount of phospho-total p 38 relativeto the hypertonic control.

As shown in FIG. 5, the polyol candidate materials, that is erythritoland xylitol reduced the amount of ERK relative to the hypertoniccontrol. The amino acids, betaine and carnitine did not.

EXAMPLE 4

Example 1 is repeated except that different concentrations of each ofthe candidate materials are used, and the TEER is measured at varioustimes from 0 to 24 hours.

Results of these tests are shown in FIG. 6. As in Example 1, the TEERvariable is represented as % TEER relative to the isotonic control.

These results demonstrate that a dose-related response was observed forL-carnitine, betaine and erythritol.

A composition including betaine and stabilized chlorine dioxide, as apreservative, was tested for component compatibility. It was found thatthe betaine was not fully compatible in such a composition. Thus,betaine is not useful with certain preservatives, such as stabilizedchlorine dioxide. However, betaine may advantageously be employed as acompatible solute in ophthalmic compositions which use otherpreservative systems, or which are free of preservatives, for example,in single or unit-dose applications.

EXAMPLE 5

Example 4 was repeated except that compositions including combinationsof compatible solutes were used. Compositions including only glycerol asa compatible solute were also tested.

Test results are shown in FIGS. 7 and 8.

These test results demonstrate that combinations of different compatiblesolutes may potentially yield added benefits.

EXAMPLE 6

The pro-piece of Major Basic Protein (MBP) has been shown to be a90-residue polypeptide.

Using established and well known techniques, a polypeptide analog of thesequence of this 90-residue polypeptide is produced.

An ophthalmic composition is prepared by blending together the followingcomponents:

Concentration % (w/v) Above-noted Polypeptide analog 0.5% Glycerol 1.0%Erythritol 0.5% Boric Acid 0.65 Sodium Borate 0.25 Sodium Citrate 0.1Potassium Chloride 0.01 Purite ®⁽¹⁾ 0.01 Sodium Hydroxide 1N Adjust pHto 7.2 Hydrochloride acid 1N Adjust pH to 7.2 Purified Water q.s. ad.⁽¹⁾Purite ® is a registered trademark of Allergan, Inc. for stabilizedchlorine dioxide. This material is added to the mixture after heatsterilization.

EXAMPLE 7

The composition of Example 6, in the form of eye drops, is administeredto the eye of a human patient about to undergo a surgical procedure inwhich the eye is to be exposed to laser energy, in particular, a LASIKsurgical procedure.

After the surgical procedure, the patient has reduced pain and/orreduced discomfort and/or reduced eye irritation and/or more rapidrecovery from the surgical procedure relative to undergoing an identicalsurgical procedure including being administered the same compositionwithout the polypeptide analog.

EXAMPLE 8

The composition of Example 6, in the form of eye drops, is administeredto the eye of a human patient undergoing a surgical procedure in whichthe eye is to be exposed to laser energy, in particular, a LASIKsurgical procedure.

After the surgical procedure, the patient has reduced pain and/orreduced discomfort and/or reduced eye irritation and/or more rapidrecovery from the surgical procedure relative to undergoing an identicalsurgical procedure including being administered the same compositionwithout the polypeptide analog.

EXAMPLE 9

The composition of Example 6, in the form of eye drops, is administeredto the eye of a human patient substantially immediately after undergoinga surgical procedure in which the eye is to be exposed to laser energy,in particular, a LASIK surgical procedure.

The patient has reduced pain and/or reduced discomfort and/or reducedeye irritation and/or more rapid recovery from the surgical procedurerelative to undergoing an identical surgical procedure including beingadministered the same composition without the polypeptide analog.

EXAMPLE 10

A series of four ophthalmic formulations in accordance with the presentinvention are prepared by blending the various components (shown in thefollowing table) together.

Concentration, % (w/v) Ingredient A B C D Carboxy 1.0 — — 0.5Methylcellulose (CMC) Glycerol 0.5 0.5 — 0.5 Erythritol 0.25 0.25 0.750.75 Boric Acid 0.60 0.60 0.60 0.60 Sodium Borate 0.045 0.045 0.0450.045 Decahydrate Calcium Chloride 0.006 0.006 0.006 0.006 DihydrateMagnesium Chloride 0.006 0.006 0.006 0.006 Hexahydrate Purite ®⁽¹⁾0.0075 0.0075 0.075 0.075 Sodium Hydroxide 1N Adjust pH Adjust pH AdjustpH Adjust pH to 7.2 to 7.2 to 7.2 to 7.2 Hydrochloric Acid 1N Adjust pHAdjust pH Adjust pH Adjust pH to 7.2 to 7.2 to 7.2 to 7.2 Purified waterq.s. ad. q.s. ad. q.s. ad. q.s. ad. ⁽¹⁾Purite ® is a registeredtrademark of Allergan, Inc. for stabilized chlorine dioxide. Thismaterial is added to the mixture after heat sterilization.

EXAMPLE 11

The procedure of Example 10 is repeated to provide the followingcompositions.

Concentration, % (w/v) Ingredient A B C D Carboxy 1.0 — — 0.5Methylcellulose (CMC) Glycerol 0.5 0.5 — 0.5 Xylitol 0.25 0.25 0.75 0.75Boric Acid 0.60 0.60 0.60 0.60 Sodium Borate 0.045 0.045 0.045 0.045Decahydrate Calcium Chloride 0.006 0.006 0.006 0.006 Dihydrate MagnesiumChloride 0.006 0.006 0.006 0.006 Hexahydrate Purite ®⁽¹⁾ 0.0075 0.00750.075 0.075 Sodium Hydroxide 1N Adjust pH Adjust pH Adjust pH Adjust pHto 7.2 to 7.2 to 7.2 to 7.2 Hydrochloric Acid 1N Adjust pH Adjust pHAdjust pH Adjust pH to 7.2 to 7.2 to 7.2 to 7.2 Purified water q.s. ad.q.s. ad. q.s. ad. q.s. ad. ⁽¹⁾Purite ® is a registered trademark ofAllergan, Inc. for stabilized chlorine dioxide. This material is addedto the mixture after heat sterilization.

EXAMPLE 12

The procedure of Example 10 is repeated to provide the followingcompositions.

Concentration, % (w/v) Ingredient A B C D Carboxy 1.0 — — 0.5Methylcellulose (CMC) Glycerol 0.5 0.5 — 0.5 Myo-inositol 0.25 0.25 0.750.75 Boric Acid 0.60 0.60 0.60 0.60 Sodium Borate 0.045 0.045 0.0450.045 Decahydrate Calcium Chloride 0.006 0.006 0.006 0.006 DihydrateMagnesium Chloride 0.006 0.006 0.006 0.006 Hexahydrate Purite ®⁽¹⁾0.0075 0.0075 0.075 0.075 Sodium Hydroxide 1N Adjust pH Adjust pH AdjustpH Adjust pH to 7.2 to 7.2 to 7.2 to 7.2 Hydrochloric Acid 1N Adjust pHAdjust pH Adjust pH Adjust pH to 7.2 to 7.2 to 7.2 to 7.2 Purified waterq.s. ad. q.s. ad. q.s. ad. q.s. ad. ⁽¹⁾Purite ® is a registeredtrademark of Allergan, Inc. for stabilized chlorine dioxide. Thismaterial is added to the mixture after heat sterilization.

EXAMPLE 13

The procedure of Example 10 is repeated to provide the followingcompositions.

Concentration, % (w/v) Ingredient A B C D Carboxy 1.0 — — 0.5Methylcellulose (CMC) Glycerol 0.5 0.5 — 0.5 Carnitine 0.25 0.25 0.750.75 Boric Acid 0.60 0.60 0.60 0.60 Sodium Borate 0.045 0.045 0.0450.045 Decahydrate Calcium Chloride 0.006 0.006 0.006 0.006 DihydrateMagnesium Chloride 0.006 0.006 0.006 0.006 Hexahydrate Purite ®⁽¹⁾0.0075 0.0075 0.075 0.075 Sodium Hydroxide 1N Adjust pH Adjust pH AdjustpH Adjust pH to 7.2 to 7.2 to 7.2 to 7.2 Hydrochloric Acid 1N Adjust pHAdjust pH Adjust pH Adjust pH to 7.2 to 7.2 to 7.2 to 7.2 Purified waterq.s. ad. q.s. ad. q.s. ad. q.s. ad. ⁽¹⁾Purite ® is a registeredtrademark of Allergan, Inc. for stabilized chlorine dioxide. Thismaterial is added to the mixture after heat sterilization.

EXAMPLE 14

The procedure of Example 10 is repeated to provide the followingcompositions.

Concentration, % (w/v) Ingredient A B C D Carboxy 1.0 — — 0.5Methylcellulose (CMC) Glycerol 0.5 0.5 — 0.5 Taurine 0.25 0.25 0.75 0.75Boric Acid 0.60 0.60 0.60 0.60 Sodium Borate 0.045 0.045 0.045 0.045Decahydrate Calcium Chloride 0.006 0.006 0.006 0.006 Dihydrate MagnesiumChloride 0.006 0.006 0.006 0.006 Hexahydrate Purite ®⁽¹⁾ 0.0075 0.00750.075 0.075 Sodium Hydroxide 1N Adjust pH Adjust pH Adjust pH Adjust pHto 7.2 to 7.2 to 7.2 to 7.2 Hydrochloric Acid 1N Adjust pH Adjust pHAdjust pH Adjust pH to 7.2 to 7.2 to 7.2 to 7.2 Purified water q.s. ad.q.s. ad. q.s. ad. q.s. ad. ⁽¹⁾Purite ® is a registered trademark ofAllergan, Inc. for stabilized chlorine dioxide. This material is addedto the mixture after heat sterilization.

EXAMPLE 15

The procedure of Example 10 is repeated to provide the followingcompositions.

Concentration, % (w/v) Ingredient A B C D Carboxy 1.0 — — 0.5Methylcellulose (CMC) Glycerol 0.5 0.5 — 0.5 Betaine⁽²⁾ 0.25 0.25 0.750.75 Boric Acid 0.60 0.60 0.60 0.60 Sodium Borate 0.045 0.045 0.0450.045 Decahydrate Calcium Chloride 0.006 0.006 0.006 0.006 DihydrateMagnesium Chloride 0.006 0.006 0.006 0.006 Hexahydrate Purite ®⁽¹⁾0.0075 0.0075 0.075 0.075 Sodium Hydroxide 1N Adjust pH Adjust pH AdjustpH Adjust pH to 7.2 to 7.2 to 7.2 to 7.2 Hydrochloric Acid 1N Adjust pHAdjust pH Adjust pH Adjust pH to 7.2 to 7.2 to 7.2 to 7.2 Purified waterq.s. ad. q.s. ad. q.s. ad q.s. ad. ⁽¹⁾Purite ® is a registered trademarkof Allergan, Inc. for stabilized chlorine dioxide. This material isadded to the mixture after heat sterilization. ⁽²⁾Betaine is found to beincompatible with the Purite7 preservative. Therefore, no preservativeis used. These compositions are useful in single or unit doseapplications.

EXAMPLE 16

The procedure of Example 10 is repeated to provide the followingcompositions.

Concentration, % (w/v) Ingredient A B C D Carboxy 0.5 — 0.5⁽³⁾ —Methylcellulose (CMC) Glycerol 0.9 0.9 0.9 0.9 Erythritol 0.5 0.5 0.250.25 Carnitine HCL 0.1 0.25 0.1 0.25 Boric Acid 0.45 0.45 0.45 0.45Sodium Borate 0.46 0.46 0.46 0.46 Sodium Citrate 0.1 0.1 0.1 0.1Potassium Chloride 0.14 0.14 0.14 0.14 Calcium Chloride 0.006 0.0060.006 0.006 Magnesium Chloride 0.006 0.006 0.006 0.006 Purite ®⁽¹⁾ 0.010.01 0.01 0.01 Sodium Hydroxide 1N Adjust pH Adjust pH Adjust pH AdjustpH to 7.2 to 7.2 to 7.2 to 7.2 Hydrochloric Acid 1N Adjust pH Adjust pHAdjust pH Adjust pH to 7.2 to 7.2 to 7.2 to 7.2 Purified water q.s. ad.q.s. ad. q.s. ad q.s. ad. ⁽¹⁾Purite ® is a registered trademark ofAllergan, Inc. for stabilized chlorine dioxide. This material is addedto the mixture after heat sterilization. ⁽³⁾A mixture of 10% by weighthigh molecular weight carboxylmethyl cellulose having a weight averagemolecular weight of about 700,000, and 90% by weight medium molecularweight carboxymethyl cellulose having a weight average molecular weightof about 250,000.

EXAMPLE 17

Each of the compositions produced in Examples 10 through 16, in the formof eye drops, is administered once a day or more often to the eyes of apatient suffering from dry eye syndrome. Administration may be either inresponse to or in anticipation of exposure to adverse environmentalconditions for example dry or windy environments, low humidity,extensive computer use, and the like. Such administration issubstantially similar to that used with conventional artificial tearcompositions.

All of the patients, after one week of such administration, are found tohave received substantial relief, for example, in terms of reduced painand/or reduced irritation and/or enhanced vision and/or enhanced eyeappearance, from the effects or symptoms of dry eye syndrome. Inaddition, those patients who are administered compositions includingcarboxymethyl cellulose (CMC) are found to have benefited from theanionic character of the CMC and the relatively increased viscosities ofsuch compositions. Such benefits include, without limitation, reducedirritation for longer periods of time after administration, and/orenhanced eye lubrication and/or enhanced protection against adverseeffects of cationic species on the ocular surfaces of the patient'seyes.

EXAMPLE 18

Each of the compositions produced in Examples 10 through 16 includingcarboxymethyl cellulose (CMC), in the form of eye drops, is administeredto an eye of a different human patient about to undergo a LASIK surgicalprocedure.

After the surgical procedure, each of the patients has reduced painand/or reduced discomfort and/or reduced eye irritation and/or morerapid recovery from the surgical procedure relative to undergoing anidentical surgical procedure including being administered the samecomposition without the carboxymethyl cellulose.

EXAMPLE 19

Each of the compositions produced in Examples 10 through 16 includingcarboxymethyl cellulose, in the form of eye drops, is administered tothe eye of a different human patient undergoing a LASIK surgicalprocedure.

After the surgical procedure, each of the patients has reduced painand/or reduced discomfort and/or reduced eye irritation and/or morerapid recovery from the surgical procedure relative to undergoing anidentical surgical procedure including being administered the samecomposition without the carboxymethyl cellulose.

EXAMPLE 20

Each of the compositions produced in Examples 10 through 16 includingcarboxymethyl cellulose, in the form of eye drops, is administered tothe eye of a different human patient substantially immediately afterundergoing a LASIK surgical procedure.

Each patient has reduced pain and/or reduced discomfort and/or reducedeye irritation and/or more rapid recovery from the surgical procedurerelative to undergoing an identical surgical procedure including beingadministered the same composition without the carboxymethyl cellulose.

While this invention has been described with respect to various specificexamples and embodiments, it is to be understood that the invention isnot limited thereto and that it can be variously practiced within thescope of the following claims.

What is claimed is:
 1. A topical ophthalmic composition for thetreatment of dry eye comprising: water; a tonicity component selectedfrom erythritol, carnitine, isomers thereof, and suitable salts thereof;carboxymethyl cellulose or its suitable salt, or a combination ofcarboxymethylcellulose or its suitable salt and hyaluronic acid; andglycerol; wherein the composition is effective for the treatment of dryeye.
 2. The composition of claim 1, further comprising a buffer selectedfrom the group consisting of sodium citrate and sodium borate, and asalt selected from the group consisting of potassium chloride, calciumchloride, and magnesium chloride.
 3. The composition of claim 1, whereinthe carboxymethly cellulose is present in an amount of about 0.5 toabout 1.0% w/v.
 4. The composition of claim 1, wherein erythritol, itsisomer, and/or its suitable salt is present in an amount of about 0.05%to about 1% w/v.
 5. The composition of claim 1, wherein carnitine, itsisomer, and/or its suitable salt is present in an amount of about 0.05%to about 1% w/v.
 6. The composition of claim 1, wherein thecarboxymethylcellulose or its suitable salt or combination ofcarboxymethylcellulose or its suitable salt and hyaluronic acid ispresent in an amount between about 0.4% w/v to about 1.3% w/v.
 7. Thecomposition of claim 1, wherein the hyaluronic acid is present in anamount of about 0.1% w/v.